Human embryonic stem cells (hESCs) present a potentially unlimited supply of cells that may be directed to differentiate into all cell types within the body and used in regenerative medicine for tissue and cell replacement therapies. An area of particular interest is stem cell transplantation for bone tissue regeneration where hESCs may be used to repair skeletal defects. Current techniques used for bone tissue repair employ the use of auto- and allografting methods, however, these methods have inherent limitations that restrict their universal application. The limitations of these reparative strategies suggest that an alternative approach is required, and hESCs may provide a repository of cells for such an approach. One of the major gaps in the knowledge regarding hESCs is the lack of understanding of the growth factors and three-dimensional signals that control differentiation. Previous work has demonstrated that hESCs can be directed to osteoblasts, however, how to achieve directed differentiation still remains a pivotal question that is unanswered. Therefore, we hypothesize that controlling the local in vitro and in vivo microenvironments can control osteoblast differentiation of hESCs. Here, we investigate the importance of cell culture conditions in-the in vitro microenvironment, and the importance of scaffold design in the in vivo microenvironment. We will begin by developing two hESC co-culture systems to direct osteoblast differentiation via pro-osteogenic factors derived from both hBMSCs and mesenchymal cells derived from hESCs (ES-MSCs). Then, we will identify a homogenous population of osteoprogenitor hESCs using the lineage specific Col2.3-GFP transgene, fluorescence-activated cell sorting (FACS) and microarray analysis. Next, we will deliver the osteoprogenitor hESCs in vivo within scaffolds to investigate the effects pro-osteogenic factor incorporation and scaffold permeability have on cell differentiation and overall bone tissue formation via micro-CT. PUBLIC HEALTH RELEVANCE: The long-term goal of this research is to further understand the biology of human embryonic stem cell development and more specifically, provide information about the organogenesis of bone. Through this research, I hope to contribute to the biomedical field in an effort to aid in the development of new hESC-based tissue engineering and regenerative medicine strategies.